382 madkor, cheese ripening components in cheese samples. the

382 Madkor Cheese ripening

components in cheese samples The results for Manshy (13) IMHOF R ISOLINI D BOSSET JO Lebensm

chego cheese obtained here can be considered as WissenschaftTechnol 23305-311 (1990)

complementary to those obtained previously with SDE (14) DUNN HC LINDSAY RC J Dairy Sci 68 2853shythe information obtained deals with highly volatile comshy 2858(1985)

pounds many of which are probably relevant for flashy (15) BADINGS HT JONG C DE DOOPER RPM J

vour The main differences between the results found High Resol Chromatogr 8 755-763 (1985)

by the present method and by SDE can be explained in (16) LELAND JV REINECCIUS GA LAHIFF M J

terms of properties such volatility and water solubility Dairy Sci 70 524-533 (1987) (17) URBACHGAustrJDairyTechnol4580-85(1990) (18) RAFECAS M BOATELLA J TORRE MC DE LA

Acknowledgement Rev Agroquim Tecnol Aliment 26 597-601 (1986)

This work was supported by project FAIR-CT97-3173 (19) BOSSET JO GAUSCH R Int Dairy J 3 359-377

from the Commission of European Communities (1993) 20) COLLIN S OSMAN M DELCAMBRE S ELshy

ZAYAT Al DUFOUR JP J Agric Food Chern 415 References 1659-1663 ( 1993)

(1) MARCOS A In Cheese Chemistry Physics and (21) YANG WT MIN DB J Food Sci 59 1309-1312 Microbiology (Ed PF Fox) Vol 2 Elsevier Applied (1994) Sci~nce London 185-219 (1987) 22) ESTEBAN JL MARTINEZ-CASTRO 1 SANZ J

(2) NUNEZ M MEDINA MGAYA PJ Dairy Res 56 J Chromatogr A 657155-164 (1992) 303-321 (1989) (23) VALERO E MIRANDA E SANZ J MARTINEZshy

(3) CHARRO A SIMAL J CREUS JM TRIGUEROS CASTRO 1 Chromatographia 44 59-65 (1997) J An Bromatol 21 7-27 (1969) (24) ESTEBAN JL VALERO E MIRANDA E JIMEshy

(4) WIROTAMA IPG NEY KH FREITAG WG Z NEZ Ml MARTINEZ-CASTRO I SANZ J MO~ Lebensm Unters Forsch 15378-82 (1973) RALES R LC-GC 15 264-2751997)

(5) ORIA R CONDON S SALA T Milchwissenschaft (25) NISTEPNNIH Mass Spectral Database PC Vers 42713-716(1987) 40 The NIST Mass Spectrometry Data Center Notshy

(6) MARTINEZ-CASTRO 1 AMIGO L SANZ J RAshy tingham UK(1992) MOS M J Dairy Res 58 239-246 (1991) (26) HA JK LINDSAY RC J Food Sci 561241shy

(7) WONG NP PARKS OW J Dairy Sci 51 1768- 1247 1250(1991) 1769(1968) (27) JANDA V PEHAL F HRIVNAK J J High Resol

(B) MANNING DJ ROBINSON HM J Dairy Res 40 Chromatogr 7 54D-41 (1984) 63-75 (1973) (28) URBACH G in Microbiology and biochemistry of

(9) FRUTOS M DE SANZ J MARTINEZ-CASTRO 1 cheese and fermented milk (D A Law) Chapman amp

Chromatographia 25 861-864 (1988) middot Hall London (1997) (10) HILD J Milchwissenschaft34281 (1979) (29) DUMONT JP ADDA J J Agric Food Chern 26

(11) HORWOOD JH Austral J Dairy Techno 44 91 364-367 (1987) (1989) (30) VIALLON C VERDIER-METZ 1 DENOYER C

(12) FERNANDEZ E J Agric Food Chern 44 1833- PRADEL P COULON JB BERDAGUE JL J 1839 (1996) Dairy Res 66 319-326 (1999) middot

Evaluation of commercial adjuncts for use in cheese middotripening 5 Effect of added freeze-shocked adjunct lactobacilli on proteolysis and sensory quality of reduced fat Cheddar cheese

By SA MADKOR1 PS TONG2 and M EL SODA3

1Department of Dairy Technology Faculty of Agriculture Minia University EI-Minia Egypt

2Dairy Products Technology Center California Polytechnic State University San Luis Obispo CA

3Department of Dairy Science and Techf1ology UniversityofAlexandria Egypt

Reduced fat Cheddar cheese curds were made using Lactococcus starter and conventional milled curd cheeseshy

making procedure After milling the curds were inoculated with no (control) or one of 6 freeze-shocked adjunct cuJtures

(-107 cfugram) of lactobacilli (4 strains of Lb helveticus I U M and Lor2 strains of Lb caseiTand A) All adjunct-treated middot

cheese had slightly higher levels of WSN than control Adjunct treated cheeses reached higher levels of FAA than control

with the progress of ripening Greater increase of totalFAA was observed in cheese inoculated with Lb helveticusl folshy

lowed by strain U and M Organoleptic evaluation indicated that cheese inoculated with Lb helveticus I rapidly developed

the highest flavor score after 3 months of ripening compared tomiddotcontrol cheese Inoculation of Lb h~vetius strains in the

cheese curds positively influenced flavor andtexture attributes in reduced fat Cheddar by preventing bitterness and reshy

ducing firmness Less influence on proteolysis and flavor intensity was observed in cheese inoculated with adjunct culshy

tures with poor rate of autolysis and lowpeptidolytic activity suchas Lb caseiT and A strains middot middot middot middot

56 Cheese ripening (commercial adjuncts) middot middot middot 56 Kasereifung (Mikrobenzusatze)

Milchwissenschaft 55 (7) 2000

Madkor Cheese ripening

1 Introduction Adjunct cultures of mesopllilic lactobacilli are used to

improve and accelerate flavor development during cheese ripening (1 4 8 22) They may play an important role as a peptidolytic agent to prevent bitterness during Cheddar cheese ripening (5 6 8 9)

Species studied as adjunct cultures for cheese inshyclude Lactobacillus plantarum Lb casei Lb helvetishycus Lb brevis and to a lesser extent Pedicoccus or Mishycrococcus (14 16 18 25) These organisms contribute to an increase in peptidase activity 1n cheese and enshyhance the production of free amino nitrogen during secondary and tertiary ripening (5 18 22) Lactobacilshylus casei and Lb heveticus have been suggested as adjunct cultures with good potential especially in reshyduced fat cheese (8 17) However positive results for flavor and texture improvement in cheese depends strongly on the strainmiddot used (11 14 28) The evidence that adjunct lactobacilli appear to have significant imshypact on Cheddar cheese maturation has encouraged culture research to select adjunct strains with approprishyate proteolytic characteristics for cheese ripening (9 11 17) Practically bacterial strains to be used as adjunct cultures in reduced fat cheese should be carefully seshylected on the basis of their temperature sensitivity autolytic properties and proteolyticpeptidolytic activishyties (9 10 23) In some reports specific catabolic activshyity of adjunct cultures may also be important (58 16) In our previous studies (12 23) several strains of laborashytory and commercially available cultures of lactobacilli varied con~iderably in their enzymatic activities and their resistance to autolytic process either in buffer sysshytem or model cheese slurry system Cheese slurries made with adjunct cultures selected for their high enshyzymatic potentials and high autolysis rate exhibited the highest levels of intracellular enzyme release and flavor development in cheese slurries Other results from our laboratory (13 24) also indicated that freeze shocked lactobacilli showed the highest rate of cell autolysis and enzyme release in model cheese slurry system comshypared to untreated cells or other physical treatments

In this study we compare the effectiveness ofmiddot seshylected strains of freeze shocked adjunct cultures of lacshy

tobacilli on the proteolysis and sensory attributes during ripening of reduced fat Cheddar cheese

2 Materials and methods

middot 2 1 Culture strains and freeze shocking treatment

middot Six commercial and laboratory culture strains of adshyjunct lactobacilli (4 strains of Lb helveticus I U M L and 2 strains Lb casei T and A) were selected accordshying to their enzymat_ic activities and autolytic properties as previously described (23) Cultures were cultivated in MRS broth at 3rC and cells were harvested by censhytrifugation at 1500 g for 30 min at 4 oc The cell pellet

-was washed twice with 001 M phosphate buffer (pH 70) and resuspended in the same buffer to obtain vishy

able cell concentrations of approximately 1 09 cfuml The cell suspension was frozen at -20degC for 24 hand

bull t~aWed iri a water bath at 40 oc prior to use _- - -

22 middot Chfesemanufacturing middot

gtmiddotjfeduc~d fat Cheddar cheeses were made from stanshymiddotbull middotdardized (proteinfat ratio -160) pasteurized (77 3 oc

middot Milchwissenschaft 55 (7) 2000

383

for 16 s) milk in 2000 liter vat according to a convenshytional protocol for milled curd Cheddar cheese-making (19) A commercial direct to vat set mesophilic factoshycoccus starter culture (type DVS 850 Chr Hansen Lab Milwaukeemiddot WI) was added to milk (0015 ww) and ripened for 45 min at 30 oc After renneting (Chymax Chr Hansen Lab Milwaukee WI 009 vw) the coshyagulum was cut and cooked to 39 oc over 30 min and held at this temperature for an additional 30 min stir out After whey drainage the curds were cheddared until the pH reached _54 and milled NaCI (17 ww) was added to the curd in 3 equal applications over 15 min Portions of the cheese curd (5 kg) were individually inshyoculated with no adjunct (control cheese) or with one of 6 freeze shocked culture suspensions of adjunct lactoshybacilli ( -107 cfug cheese) While cultures are normally added to the cheese milk adjunct cultures were added directly to cheese curd in this study to insure equal numbers of adjunct culture per gram of cheese curd The cheese curd were mixed thoroughly to ensure even distribution of culture cells hooped in rectangular blocks pressed at 40 psi for 3 h vacuum packaged and ripshyened at 1 0 oc for 6 months

23 Chemicalandmicrobiological analysis of cheese

Duplicate samples of cheese were analyzed for moisture fat salt total protein and pH (3) Proteolysis was assessed by measuring water-soluble nitrogen (20) total free amino acids using the cd-ninhydrin method (15) and urea-PAGE (2) The counts of viable cells of lactobacilli in cheese were determined by enumeration on MRS plate agar and incubation at 32 oc for 72 h

24 Sensory evaluation

Cheese samples were graded for flavoraroma and bodytexture at 3 and 6 month by 5 experienced Chedshydar cheese taste panelists on a 1-10-point scale with more than 70 indicating high quality Cheddar cheese flavor or texture and less than 4 indicating poor quality Cheddar cheese flavor or texture (24) Panelists comshymented on Cheddar flavor intensity bitterness off-flavor and firmness

3 Results and discussion 3 1 Compositional andmicrobiological analysis

The composition of the 2 trials were found to be within an average for moisture fat protein and salt of 4365plusmn115 176plusmn030 2985plusmn082 and 155plusmn008 respectively One-day old cheese displayed an avershyage pH of 518plusmn006 In fact the experimental cheese pHs were not affected by inoculation of adjunct lactoshybacilli which may due to the low residual lactose in curd or the low ability of the added freeze shocked adjunct lactobacilli to maintain activity to ferment the residual lactose and produce acid (14 21 )

In both trials the initial counts of non-starter lactobashycilli(Fig 1) in the experimental cheese after one day of ripening were between 1 Q4-1 os cfug cheese The numshyber is lower than the number added to cheese curd possibly due to the loss of some adjunct cells when

mixed with cheese curd losses due to pressing and

384

loss of viability Very low numbers of non-starter lactoshybacilli were detected on MRS media of control cheese (lt102 cfug) This number of advantageous non-starter lactobacilli in control cheese reached a level of -1 04

after 3 months of ripening and remained constant up to the end of ripening However the non-starter lactobashycilli counts in the experimental cheese were most likely those recovered from inoculation of adjunct cultures in cheese curd Cheese inoculated with Lb casei strain showed dramatic increases in the number of lactobacilli reaching 1 Q9 cfug cheese after about 3 month of ripenshying and then slightly 1iecreased at the end of ripening The corresponding number of viable cells retained in cheese with Lb helveticus strains was between 1QL1os after 3 months of ripening and decreased with the proshygress of ripening A decrease in cell counts of adjunct lactobacilli after 2-6 month of ripening was also noted in several studies (17 21 22) which may have been due to autolysis of the adjunct cells (14) The decreasing rate of cell viability was more pronounced in some cheese In particular those inoculated with Lb hevetishycus I had a high extent of cell lysis (14 23) Reported data in our proceeding work (13 26) demonstrated that cell lysis is strain specific and markedly enhanced by freeze shocking treatment

10

9 CD Ul 8CD CDs 7 0

~ 6 -sect 5

0

Oi 40 I

3

2

30 90 180

Ripening time (days)

Fig 1 Counts of viable lactobacilli during ripening of reshyduced-fat Cheddar cheese made without adjunct (control cheese +) or with added freeze-shocked adjunct cultures of Lb helveticus I (bull) U (amp) M (bull) and L (D) or Lb caseiT ()and A (0)

32 Proteolysis

Proteoysis as assessed by the amount of WSN (Fig 2) increased linearly with time for all Cheese up to the end of ripening period All adjunct treated cheeses showed somewhat slightly higher levels of WSN than control This trend was manifested more in Lb hevetishycus strains I when compared to Lb casei Also the proshyteolytic activity of Lb helveticus I may have also been responsible for sorrie qualitative differences in peptide profiles which were detected in gel electrophoretoshygrams of 3 and 6 month old cheese (Fig 3)

As in our preceding report 24) the levels of total free amino acids (FAA) was significantly affected by adjunct cultures The corresponding levels of total FAA as measshy

middotured by the cd-ninhydrin method (Fig 4) reached conshysiderably higher levels of FAA than control There were


only slight differences in the levels of FAA between all cheeses up through 2 month of ripening however higher concentration of total FAA developed thereafte~ in adjunct treated cheeses than control cheese through the end of ripening A greater increase in the level of total FAA was observed in cheese inoculated with Lb heveticus I Notably in cheese inoculated with Lb helshyveticus I the total free amino acids increased considshyerably with time and was higher than for all other strains at 6 month of ripening It is interesting to notice that the level of FAA in 3-month old cheese inoculated with Lb helveticus I reached a value similar to that in 6-month old cheese without adjunct

28

t (]) 24Ol e g s 20

s 160

~ 0

Ul l1l 12 z rJ)

8~

30 60 90 120 180


Fig2 Formation of water-soluble nitrogen (WSN) during rjpening of reduced-fat Cheddar cheese made withshyout adjunct (control cheese+) or with added freezeshyshocked adjunct cultures of Lb heveticus I (bull) U (A) M (e) and L (D) or Lb caseiT ()and A (0)

3 Months 6 Months

c l1 2 3 4 s 6 1l Ia 9 10 11 12 13 141

y-Casein

~-Casein

as1-Casein

Fig 3 Gel electrophoretograms of Cheddar cheese at 3 (lane 1-7) and 6 (lane 8-14) month of ripening inocushylated with freeze shocked adjunct strains of Lb heshyveticus I (lane 2 9) M (lane 3 10) U (lane 4 11) L middot (lane 6 13) or Lb caseiT (lane 5 12) and A (7 14) Control (no adjunct lane 1 8) Na-caseinate (lane C)

Other strains of Lb helveticus have been reported to contribute to the levels of FAA in reduced fat Cheddar cheese (8 17) The data revealed that all adjunct culshytures evaluated herein would influence development of FAA but to different degree Consequently substantial increase in free amino group content of cbeese m~ade with Lb helv~ticus I strain may have been due to their highpeptidolytic activity and increased celllysis In our

Milchwissenschaft 55 (7) 2000 middot


pre~ding studies (12 13 23) certain strains of lactoshybacrlh such as Lb caseiT and A were expected to have less influence in promoting proteolysis due to their low rate of enzyme release and proteolytic activity Thus depending on strain type the extent of enzyme release and the effectiveness of peptidase activities of $elected lactobacilli added to cheese the WSN peptides can be degraded extensively to elevated levels of FAA (5 8 22 24) In fact enhanced cell lysis and high aminopepshytidase activities of selected lactobacilli can be a limiting factor in the rapid formation of flavor constituents during ripening of Cheddar cheese with reduced fat content A strong correlation between the extent of aminopeptidoshylysis and cheese flavor intensity was revealed in sevshyeral other reports (6 8 21 25)

10

~ Ill CD 8CD c 0

~ Cl CD 6 t ti ~

~ Cl 4 sect lt( lt( lL 2

30 60 90 120 180


Fig 4 Development oftotal free amino acids during ripening of reduced-fat Cheddar cheese made without adjunct (control cheese +) or with added freeze-shocked adshyjunct cultures of Lb helveticus I (11) U (A) M(bull) and L(D)orLbcaseiT()andA(O)


The sensory score of cheese (Fig 5) indicates that highest scores for flavor and texture were received for cneese inoculated with Lb helveticus I followed by Lb helveticus M and U Taste panelists noted that adjunct treated cheese in particular cheese inoculated with Lb helveticus I rapidly developed the highest flavor score after 3 month of ripening compared to control cheese Cheeses treated with some adjuncts such as Lb casei T orA were downgraded beQause the panelist indicated that the c~eese contained a sour-milky taste which reshym~ined constant _in these cheeses up to the end of ripshyen~ng The panelist comments also indicated that conshytrol cheese laCked flavor after 3 month of ripening and with extended ripening tended to have bitterness Flashyvor scores in cheese made with Lb helveticus 1was highest at 3 and 6 month of ripening period Other reshyported studies revealed that adjunct cultures of Lb helshyl(etitiJs p9sitiv_ely influenced flavor and prevented bitshy

terness during ripening of reduced fat Cheddar cheese through their high amino peptidolytic activities (8 17 28)

Cheeses inoculated with Lb helveticus I and M middot t~ndedtb ~ave higher texture scores relatively softer body andJ~ssfrrtnness compared to control The higher proshy

teqlys1s rates of some adjunct treated cheese evaluated

Milchwssenschaft 55 (7) 2000

385

herein may be associated with the decrease in firmness particularly in low fat cheese (8) Enhanced breakdown of the casein matrix especially a-casein has been associshyated with improved texture and smooth body in reduced fat cheese (8 7 28) However uncontrolled proteolysis can negatively impact cheese body and machinability

9~------------~--------------~ Flavor amp Aroma Texture amp Body

8

7

~ 6 0 0 Ill 5 CD Ill

3l 4 c u 3

2

I U M L T A e I U M L T A c 0 u

Fig 5 Sensory scores of 3 (D) and 6 (11) month old reducedshyfat Cheddar cheese made without adjunct (control cheese) or with added freeze-shocked adjunct culshytures of Lb helveticus I U Mand Lor Lb caseiTand A

4 Conclusion The present study confirms and expands upon the

findings of our previous studies (23 24) that freeze shocked Lb helveticus in particular strain I can be used successfully to enhance flavor development and imshyprove quality of reduced fat Cheddar cheese The addishytion of freeze shocked Lb heveticus I has the important advantage of supplementing whole cell enzyme system which increases the proteolytic potential in cheese Inshyoculation of Lb helveticus with high autolytic properties and high peptidolytic activity led to substantial increase in tertiary proteolysis rate resulting in elevated levels of small peptides and free amino acids Consequently cheese maturity and quality were enhanced as detershymined by the high flavor score after 3 and 6 month of ripening Also Lb helveticus I positively influenced flashyvor and texture attributes in reduced fat Cheddar by preventing bitterness and reducing firmness Less in- middot fluence on proteolysis and flavor intensity were obshyserved when adjunct cultures with low rates of autolysis and low peptidolytic activity was used (eg Lb casei T and A strains)

Acknowledgements

Research financial support by Dairy Management Inc and California Dairy Research Foundation is gratefully acknowledged We thank Jerry Mattas and Sean Vink for the expert technical assistance in cheese-making and analytical work

5 References (1) ALY M Nahrung34329-335(1990) (2) ANDREWS AT J Dairy Res 5045-55 (1983) (3) APHA Standard Methods for Examination Dairy Prod

15thedAm Publ Health AssocWashington DC (1985)

386 Halbert Wheyproteins

(4) BARTELS H JOHNSON M OLSON N Milchshywissenschaft 42 139-144 ( 1987)

(5) BROADBENT J Proc 11th Biennial Cheese Cont Logan UT August (1994)

(6) BROOME MC KRAUSE DA HICKEY MW AUST J Dairy Techno 45 67-73 (1990)

(7) CREAMER LK OLSON NF J Dairy Sci 47 631shy635 (1982)

(8) DRAKE MA BOYLSTON TD SPENCE KD SWANSON BG Food Res Int 30 35-40 (1997)

(9) El ABBOUD M EL SODA M PANDIAN S BARREAU MTREPANIER G SIMARD RE Int Dairy J 2 55-64 (1991)

(10) EL KHOLY W EL SODA M EZZAT N ELshySHAFEI H Lait781-14(1998)

(11) EL SODA M In Microbiology and Biochemistry of cheese and Fermented Milk (Ed B Law) Blackie Acad amp Profess London NewYork219-246 (1997)

(12) EL SODA M MADKOR SA TONG PS Milchshywissenschaft 54 85-89 (1999)


(14) ELSODA M MADKOR SA TONG PS J Dairy Sci 83 609-619 (2000) middot

(15) FOLKERTSMA B FOX PF J Dairy Res 59217shy224 (1992)

(16) FOX PF McSWEENEY PLH LYNCH CM Aust J DairyTechnol53 83-89 (1998)

(17) JOHNSON J ETZEL M CHEN C JOHNSON M J Dairy Sci 78 769-776 (1995)

(18) KHALID NM MARTH EH J Dairy Sci 73 2669shy2684 (1990)

(19) KOSIKOWSKI F In Cheese and Fermented Milk Foods Edwards Brothers Arbor Ml 228-260 (1977)

(20) KUCHROO CN FOX PF Milchwissenschaft 37middot 331-335 (1982)

(21) LYNCH C M McSWEENEY PLH FOX PF COGAN TM DRINAN FD Int Dairy J 6 851shy867(1996)

(22) LYNCH CM MUIR DD BANKS JM McSWEEshyNEY PLH FOX PF J Dairy Sci 82 1618-1628 (1999)

(23) MADKOR SA EL SODA M TONG PS Milchshywissenschaft 54133-137 (1999)

(24) MADKOR SA TONG PS EL SODA M J Dairy Sci (accepted for publication)

(25) PETERSON SD MARSHALL RT J Dairy Sci 731395-1410(1990)

(26) THIBOUTOT H DAKO E EL SODA M VUILshyLEMARD JC POWER N SIMARD RE Milchshywissenschaft 50 448--452 ( 1995) middot

(27) VISSER S J DairymiddotSci76 329-350 (1993) (28) YARDO Y In Microbiology and Biochemistry of

Cheese and Fermented Milk Ed BA Law Blackie middot Academicamp Professional207-218 (1997)

Stabilisation of calcium phosphate using denatured whey proteins

By C HALBERT1 BTOKENNEDY2 A HALLIHAN1 and PM KELLY2

1Food Technology Dept University College Cork Ireland 2Dairy Products Research Centre Teagasc Moorepark Fermoy Co Cork Ireland

Denatured whey proteins (3 ww) were shown to stabilise 30 mM calcium and phosphate Subsequent stability to heat was dependent on the initial pH of the calcium phosphatewhey protein mixture At64 lt pH lt 67 stand-up gels were formed on heating to 90degC for 30 min At pH gt67 no gelation was evident The degree of unfoldingaggregation of whey protein prior to addition of calcium and phosphate had a significant effect on the strength of the gel observ~d after subseshyquent heating The duration of heating at 70 oc showed major effects on gel strength following calcium and phosphate addition at pH 65 and subsequent re-heating The strength of the gels following pre-heating at 80 oc was also affected by duration of heating but less so than at 70 oc Pre-heating at 90 oc resulted in gels which decreased in strength with inshycreased duration of heating The strength of themiddotgels formed from whey protein which had been pre-heated at 90 oc esmiddot pecially for the longer times were weaker than gels formed from the 80 oc pre-heat treatment The secondary hea~lng step was shown to induce a major release of protons due to secondary calcium phosphate formation and the accompanyshying decrease in pH aided in gel formation middot middot

66 Whey proteins (phosphate stabilisation) 66 Molkenproteine (Phosphatstabilisierung)

1 Introduction Whey proteins are globular proteins and are denashy subsequent aggregation of the protein molecules (5)

tured by heat They are widely used to enhance the nu~ Any action that changes the native conformation of the tritional value of formulated foods and are often cited as protein has in principle the potential to induce gelashybeing highly functional due to their ability to gel on heat- middot tion This may include heat treatment (6) addition of ing and immobilise large quantities of water (1 2 3) calcium sodium or acid (7 8 9) or enzymatic treatment Whey proteins can form either thermo-reversible or (1 0 11 ) A major force which opposesmiddotprotein aggrega- middot thermo-irreversible gels when heated above 65 to 70degC tionis electrostatic repulsion between similarly charged

depending on the initial pH and composition of the pro~ protein molecules (12) therefore protein aggregaton is tehsolution ( 4) Thermal gelation of whey proteins is a particularly sensitive to pH the ionic species present and two-stage process involving an initial unfolding and a ionic strength In this study the possibility ot introducing



1 Introduction Adjunct cultures of mesopllilic lactobacilli are used to

improve and accelerate flavor development during cheese ripening (1 4 8 22) They may play an important role as a peptidolytic agent to prevent bitterness during Cheddar cheese ripening (5 6 8 9)

Species studied as adjunct cultures for cheese inshyclude Lactobacillus plantarum Lb casei Lb helvetishycus Lb brevis and to a lesser extent Pedicoccus or Mishycrococcus (14 16 18 25) These organisms contribute to an increase in peptidase activity 1n cheese and enshyhance the production of free amino nitrogen during secondary and tertiary ripening (5 18 22) Lactobacilshylus casei and Lb heveticus have been suggested as adjunct cultures with good potential especially in reshyduced fat cheese (8 17) However positive results for flavor and texture improvement in cheese depends strongly on the strainmiddot used (11 14 28) The evidence that adjunct lactobacilli appear to have significant imshypact on Cheddar cheese maturation has encouraged culture research to select adjunct strains with approprishyate proteolytic characteristics for cheese ripening (9 11 17) Practically bacterial strains to be used as adjunct cultures in reduced fat cheese should be carefully seshylected on the basis of their temperature sensitivity autolytic properties and proteolyticpeptidolytic activishyties (9 10 23) In some reports specific catabolic activshyity of adjunct cultures may also be important (58 16) In our previous studies (12 23) several strains of laborashytory and commercially available cultures of lactobacilli varied con~iderably in their enzymatic activities and their resistance to autolytic process either in buffer sysshytem or model cheese slurry system Cheese slurries made with adjunct cultures selected for their high enshyzymatic potentials and high autolysis rate exhibited the highest levels of intracellular enzyme release and flavor development in cheese slurries Other results from our laboratory (13 24) also indicated that freeze shocked lactobacilli showed the highest rate of cell autolysis and enzyme release in model cheese slurry system comshypared to untreated cells or other physical treatments

In this study we compare the effectiveness ofmiddot seshylected strains of freeze shocked adjunct cultures of lacshy

tobacilli on the proteolysis and sensory attributes during ripening of reduced fat Cheddar cheese

2 Materials and methods

middot 2 1 Culture strains and freeze shocking treatment

middot Six commercial and laboratory culture strains of adshyjunct lactobacilli (4 strains of Lb helveticus I U M L and 2 strains Lb casei T and A) were selected accordshying to their enzymat_ic activities and autolytic properties as previously described (23) Cultures were cultivated in MRS broth at 3rC and cells were harvested by censhytrifugation at 1500 g for 30 min at 4 oc The cell pellet

-was washed twice with 001 M phosphate buffer (pH 70) and resuspended in the same buffer to obtain vishy

able cell concentrations of approximately 1 09 cfuml The cell suspension was frozen at -20degC for 24 hand

bull t~aWed iri a water bath at 40 oc prior to use _- - -

22 middot Chfesemanufacturing middot

gtmiddotjfeduc~d fat Cheddar cheeses were made from stanshymiddotbull middotdardized (proteinfat ratio -160) pasteurized (77 3 oc

middot Milchwissenschaft 55 (7) 2000

383

for 16 s) milk in 2000 liter vat according to a convenshytional protocol for milled curd Cheddar cheese-making (19) A commercial direct to vat set mesophilic factoshycoccus starter culture (type DVS 850 Chr Hansen Lab Milwaukeemiddot WI) was added to milk (0015 ww) and ripened for 45 min at 30 oc After renneting (Chymax Chr Hansen Lab Milwaukee WI 009 vw) the coshyagulum was cut and cooked to 39 oc over 30 min and held at this temperature for an additional 30 min stir out After whey drainage the curds were cheddared until the pH reached _54 and milled NaCI (17 ww) was added to the curd in 3 equal applications over 15 min Portions of the cheese curd (5 kg) were individually inshyoculated with no adjunct (control cheese) or with one of 6 freeze shocked culture suspensions of adjunct lactoshybacilli ( -107 cfug cheese) While cultures are normally added to the cheese milk adjunct cultures were added directly to cheese curd in this study to insure equal numbers of adjunct culture per gram of cheese curd The cheese curd were mixed thoroughly to ensure even distribution of culture cells hooped in rectangular blocks pressed at 40 psi for 3 h vacuum packaged and ripshyened at 1 0 oc for 6 months

23 Chemicalandmicrobiological analysis of cheese

Duplicate samples of cheese were analyzed for moisture fat salt total protein and pH (3) Proteolysis was assessed by measuring water-soluble nitrogen (20) total free amino acids using the cd-ninhydrin method (15) and urea-PAGE (2) The counts of viable cells of lactobacilli in cheese were determined by enumeration on MRS plate agar and incubation at 32 oc for 72 h


Cheese samples were graded for flavoraroma and bodytexture at 3 and 6 month by 5 experienced Chedshydar cheese taste panelists on a 1-10-point scale with more than 70 indicating high quality Cheddar cheese flavor or texture and less than 4 indicating poor quality Cheddar cheese flavor or texture (24) Panelists comshymented on Cheddar flavor intensity bitterness off-flavor and firmness

3 Results and discussion 3 1 Compositional andmicrobiological analysis

The composition of the 2 trials were found to be within an average for moisture fat protein and salt of 4365plusmn115 176plusmn030 2985plusmn082 and 155plusmn008 respectively One-day old cheese displayed an avershyage pH of 518plusmn006 In fact the experimental cheese pHs were not affected by inoculation of adjunct lactoshybacilli which may due to the low residual lactose in curd or the low ability of the added freeze shocked adjunct lactobacilli to maintain activity to ferment the residual lactose and produce acid (14 21 )

In both trials the initial counts of non-starter lactobashycilli(Fig 1) in the experimental cheese after one day of ripening were between 1 Q4-1 os cfug cheese The numshyber is lower than the number added to cheese curd possibly due to the loss of some adjunct cells when

mixed with cheese curd losses due to pressing and

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382 madkor, cheese ripening components in cheese samples. the

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